3

Aging

• 1950’s– Believed that cultivated cells could grow forever

• If not, then it was a result of a culturing deficiency

– In 1943, a cancer cell was grown in culture indefinitely

– Leonard Hayflick noticed that human fibroblasts from embryonic tissue could only grow for several months

4

Hayflick Phenomenon

• Limited replication potential of somatic cells

• 50-60 population doublings

• Stop cell cycle and enter G0 state– Senescence

5

Senescent Phenotype

Gene expression differences

Cyclin D1& D2

p21 & p16c-fos and Egr-1 Cyclins A, B, & H

Protein activity differences

SRF DNA bindingp53 and Rb activity

DNA-PKRas

PKC

Young Pre-senescent Senescent

Phenotypic differences

Large Flattened cellsUnresponsive to growth mitogens

Increase in acid β-galactosidaseIncreased excretion of extracellular matrix

Remain viable and metabolically active

6

Aging

• Late 1950’s– Cytogenetics could detect Barr Body

• Thus, distinguish male-donated fibroblasts from female-donated fibroblasts

– Thus, distinguish cells at various cell doubling stages

7

Aging

• Fibroblasts taken from young donors had a greater PDL than older doners

• Frozen cells thawed remembered their place in the PDL

• Must be some “counter”

8

Eurika!

• Harley et al – 1990– Telomeres shorten during aging of human

fibroblasts

9

Telomeres

• Telomeres– 3-20 Kb repeat of …TTAGGG…at each end of every

chromosome

• Several functions– “cap” the end of chromosomes to project against

fusion with other chromosomes– Replication– Positioning

10

Eurika!

• Harley et al – 1990– Telomeres shorten during aging of human

fibroblasts

12

Telomeres

• Telomerase• Ribonucleoprotein

• Specialized reverse-transcriptase

• Binds to 3’ overhang and synthesizes telomere repeat

13

Telomeres

• Numerous proteins bind to telomere repeats– Eg. Telomere repeat binding factor-1 and 2

(TRF1/2)

Blackburn, Cell, 2001

14

Telomeres

• Numerous proteins bind to telomere repeats– Eg. Telomere repeat binding factor-1 and 2

(TRF1/2)

• Longer repeats – more TRF1/2 binding

• Eventually inhibits telomerase activity– Thus, telomere length is restricted

15

Telomeres• In somatic cells, telomerase activity is low

• In stem cells, e.g. germ line, telomerase activity is high – maintain telomere length

• In Cancer cells, telomerase is also high

16

Telomeres

Molecular Biology of the Cell, 4th Edition, Garland Science Inc.

Telomerase knockout mice

Telomeres shorten progressively intelomerase-null mice

18

Telomeres

• Loss of telomerase activity in mice leads to premature aging

19

What happens when telomeres get too short?

• Cell detects short telomere ends and become senescent or undergo apoptosis

• Biological clock for regulating the number of cell divisions for a cell

• Genes located near telomeres may be regulated by length – age-regulated gene expression

20

Dolly the sheep

• Cloned by nuclear transfer from a 6 year old sheep.

• Telomere length 80% of normal

• Died from Infection/Cancer at age 6 (life expectancy Age 11-12)

•Chronic Arthritis at age 5

•Cloned sheep generally have shorter telomeres, but are reset in their progeny.

21

Telomeres and Human Pathology

• Werner syndrome

• Premature senescence and damage to various tissues

• Fibroblasts from Werner patients only divide about 20 times

22

Werner Syndrome

• Causative agent is mutation in WRN gene which encodes a RecQ helicase

• Mutations in WRN gene cause Werner syndrome in humans

23

Werner Syndrome

• Where does RecQ do most of its unwinding?

24

Werner Syndrome

• Forced expression of telomerase counter-acts the loss of WRN gene

• Maintenance of telomeres in humans is critical for providing genomic stability and replication potential

Hutchinson Gilford Progeria Syndrome

• rare progressive autosomal dominant disorder .

• The most striking feature of the disorder is extremely accelerated aging (progeria).

• In most cases, affected infants appear to develop normally until approximately six months of age.

•In most patients, Hutchinson-Gilford Progeria Syndrome is caused by de novo sporadic mutation in lamin A.

The Zmpste24 -/- Mouse

6 Months4 Months

Western blots of extracts from wild-type, Zmpste24–/–, and Zmpste24–/–Lmna+/– MEFs with a carboxyl (C)-terminal prelamin A antibody and an amino (N)-terminal lamin A/C antibody.

Fong L G et al. PNAS 2004;101:18111-18116

©2004 by National Academy of Sciences

Analysis of nuclear shape in wild-type, Zmpste24–/–, and Zmpste24–/–Lmna+/– MEFs by laser-scanning fluorescence microscopy.

Loren G. Fong et al. PNAS 2004;101:18111-18116

©2004 by National Academy of Sciences

Growth rates and grip strength in mice.

Fong L G et al. PNAS 2004;101:18111-18116

©2004 by National Academy of Sciences

Crossing the zmpste24 -/- with p53 -/- leads to partial rescue of the progeria phenotype.